HIV-1 latency has been identified as a major obstacle towards the development of a curative HIV-1 therapy, but previous attempts to therapeutically eradicate the latent HIV-1 reservoir have yielded only disappointing results. The problem with the reactivating agents used in some of these studies (IL-2 or anti- CD3 mAb OKT3) was that the concentrations required for efficient HIV-1 reactivation would potently trigger cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1 reactivating agents would have to trigger HIV-1 reactivation without the induction of cytokine gene expression. (1) During the drug screening effort in the first funding period we identified a protein activity from the non-pathogenic bacterium Massilia timonae that efficiently reactivated latent HIV-1 infection without triggering cellular cytokine expression (HIV-1 reactivating protein or HRF). HRF induces a short, non-sustained NF-&#954;B peak, which was insufficient to induce significant levels of cellular gene expression, but efficiently triggered HIV-1 reactivation. HRF serves as proofof- principle that dissociation of HIV-1 reactivation from the induction of cellular gene expression can be achieved by controlling the amplitude and the duration of the induced NF-&#954;B peak. We now have identified HRF using a genomic expression library and will elucidate the underlying mechanisms of HRF signaling. HRF will serve as a blueprint for the drug development effort. (2) In addition, pharmaceutical hits identified in our drug screen revealed a key role for PIM-1 kinase in the control of HIV-1 reactivation. Pharmaceutical or shRNA-mediated PIM-1 inhibition suppressed HIV-1 reactivation even in the presence of high-level NF-&#954;B activity, suggesting a gate-keeper function of the PIM-1 associated signal transduction pathway. We will now proceed to identify the molecular down-stream targets of PIM-1 that provide control at the level of the viral promoter. (3) Our data further imply that coordinated stimulation of several pathways is required to reactivate latent HIV-1 infection in the absence of cytokine gene induction. Such compound combinations would unlikely have been identified in any previous drug screen for HIV-1 reactivating agents that were designed according to the commonly applied [unreadable]one-drug-one target[unreadable] hypothesis. We here outline a concept for an iterative drug screen for activator/modulator drug combinations and propose a drug repositioning effort and the screen of a 50,000 compound library focused on kinase interacting molecules. In an initial screen used to transfer the assay to our robotic platform, we already identified 3 FDA approved drugs with potent modulator characteristics, for two of which we have identified the mechanism of action. At this time, the drug screening effort during the first funding cycle of our application has revealed three major new concepts how to target latent HIV-1 infection. In this competitive renewal we ask for the funds to develop these strategies towards possible translation into a therapeutic setting.